Xiaojuan Xu

Branching structure and chain conformation of water-soluble glucan extracted from Auricularia auricula-judae.

A water-soluble neutral polysaccharide (AF1) was extracted from Auricularia (A.) auricula-judae with 0.15 M aqueous NaCl at 80-100 °C. Its chemical components and structure were analyzed by GC, GC-MS, and NMR. AF1 was identified as a β-(1→3)-D-glucan with two β-(1→6)-D-glucosyl residues for every three main chain glucose residues, showing a comb-branched structure. The M(w) values of AF1 in both aqueous solution and DMSO determined by LLS and SEC-LLS were in the narrow range of 2.07-2.15 × 10(6), indicating AF1 existed as single chains in the two solvents. The high intrinsic viscosity [η] of 1753 mL/g and the structure-sensitive parameter ρ (≡R(g)/R(h)) value of 2.3 in water revealed that AF1 existed as stiff chain conformation. Moreover, we directly observed the extended stiff chain conformation by AFM. The branching structure led to the water solubility of AF1, and the intramolecular hydrogen bonds sustained the stiff chain conformation. The rheological results showed that this polysaccharide aqueous solution had higher viscosity than even xanthan, a pronounced thickening agent. This work provided important information for developing new thickeners in food fields, and how neutral polysaccharides can be used as good candidates.

Triple Helical Polysaccharide-Induced Good Dispersion of Silver Nanoparticles in Water

Silver nanoparticles were constructed by using triple helical polysaccharide (lentinan) dissolved in water as matrix for the first time. The structure, morphology, and size of the nanocomposites in the polysaccharide aqueous solutions were investigated with UV-visible spectroscopy (UV-vis), transmission electron microscopy (TEM), and dynamic laser light scattering (DLS). The results revealed that the silver nanoparticles were attached to the polysaccharide chains through the strong noncovalent interactions, leading to the good dispersion of silver nanoparticles with mean radius of 6 nm in water. The silver nanoparticles were stable in the lentinan aqueous solution for 9 months. However, with an addition of NaOH, the polysaccharide with the imperfect helical structure broken partially by NaOH could aggregate in the alkali aqueous solution. The aggregation of the lentinan-bonded silver nanoparticles increased with an increase in the NaOH concentration, whereas the size of the silver nanoparticles barely changed, further confirming that the Ag nanoparticles were stable in this system. The aggregation was related to the conformation transition of the polysaccharide from the triple helix to random coil in the solution. A new method to detect the aggregates and aggregation rate was established according to the intensity of the maximum absorption peaks of the polysaccharide labeled by Ag nanoparticles in the UV spectrum.

Immunomodulatory β-Glucan from Lentinus edodes Activates Mitogen-activated Protein Kinases and Nuclear Factor-κB in Murine RAW 264.7 Macrophages

Lentinan, a cell wall β-glucan from the fruiting bodies of Lentinus edodes, is well known to be a biological defense modifier, but the signal transduction pathway(s) induced by Lentinan have not been elucidated. In this study, we extracted Lentinan (LNT-S) by ultrasonication from Lentinus edodes and report that, in murine RAW 264.7 macrophages, LNT-S glucan activated NF-κB p65 and triggered its nuclear translocation as determined by Western blotting. Moreover, LNT-S enhanced NF-κB-luciferase activity in the Dual-Luciferase gene system assay. Its upstream signaling molecules, MAPKs such as ERK1/2 and JNK1/2, were shown to be activated by assessing the level of phosphorylation in a time- and concentration-dependent manner, but its downstream proinflammatory enzyme, inducible NOS, was not observed. The data evaluated using a TNF-α ELISA kit and Griess reagent further demonstrated that no proinflammatory mediators such as TNF-α and NO were produced by LNT-S stimulation in RAW 264.7 cells. In contrast, LPS significantly induced inducible NOS expression and increased NO and TNF-α production, which are associated with activation of the NF-κB p65/p50 heterodimer complex. It is possible that LNT-S did not activate NF-κB p65/p50, and the activation of NF-κB p65 was not sufficient to stimulate cytokine production. These data demonstrate that LNT-S glucan carries out its immunomodulating activity by activating MAPK signaling pathways without secretion of TNF-α and NO.

β-Glucan from Lentinus edodes inhibits nitric oxide and tumor necrosis factor-α production and phosphorylation of mitogen-activated protein kinases in lipopolysaccharide-stimulated murine RAW 264.7 macrophages.

Background: High level of NO and TNF-α can induce diverse effects on host survival. Results: Lentinan inhibits NO and TNF-α secretion and phosphorylation of MAP kinases JNK1/2 and ERK1/2. Conclusion: Inhibition of NO and TNF-α is partially through suppression of JNK1/2 and ERK1/2 activation. Significance: A novel pharmacological molecule is discovered to control the diseases associated with NO and TNF-α overproduction. Lentinan (LNT), a β-glucan from the fruiting bodies of Lentinus edodes, is well known to have immunomodulatory activity. NO and TNF-α are associated with many inflammatory diseases. In this study, we investigated the effects of LNT extracted by sonication (LNT-S) on the NO and TNF-α production in LPS-stimulated murine RAW 264.7 macrophages. The results suggested that treatment with LNT-S not only resulted in the striking inhibition of TNF-α and NO production in LPS-activated macrophage RAW 264.7 cells, but also the protein expression of inducible NOS (iNOS) and the gene expression of iNOS mRNA and TNF-α mRNA. It is surprising that LNT-S enhanced LPS-induced NF-κB p65 nuclear translocation and NF-κB luciferase activity, but severely inhibited the phosphorylation of JNK1/2 and ERK1/2. The neutralizing antibodies of anti-Dectin-1 and anti-TLR2 hardly affected the inhibition of NO production. All of these results suggested that the suppression of LPS-induced NO and TNF-α production was at least partially attributable to the inhibition of JNK1/2 and ERK1/2 activation. This work discovered a promising molecule to control the diseases associated with overproduction of NO and TNF-α.

Chain conformation and anti-tumor activity of derivatives of polysaccharide from Rhizoma Panacis Japonici

A hyper branched (1→4)-α-d-glucan (RPS3) with degree of branching of 35% extracted from Rhizoma Panacis Japonici adopted a sphere-like conformation and showed little bioactivity. Three derivatives including sulfated (S-), phosphated (P-) and carboxymethylated (CM-) RPS3 were then synthesized and characterized by FTIR, (13)CNMR, and SEC-LLS-Vis-RI. As a result, the molecular weights of CM-RPS3, S-RPS3 and P-RPS3 decreased sharply in contrast to the original one, suggesting that chemical degradation has occurred. Moreover, the sphere-like conformation of RPS3 transferred into the random coil-like conformation according to the increased values of αη, αs and ρ. It was ascribed to the occurrence of the preferential degradation for the branches. In vitro and in vivo tests demonstrated that the negatively charged S-RPS3 and P-RPS3 with properly low molecular mass significantly inhibited H-22 tumor cells growth. This work offered valuable results for broadening the biological applications of α-d-glucans.

Construction of high strength hollow fibers by self-assembly of a stiff polysaccharide with short branches in water

The development of biological high-performance materials fabricated from natural polysaccharides has attracted great attention for a sustainable world. In this work, hollow fibers with high strength were spun from a polysaccharide aqueous solution at a concentration of 0.02 g mL−1. The polysaccharide was a comb-like β-glucan with short branches isolated from Auricularia auricula-judae, coded as AF1. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed directly that AF1 existed as a stiff chain conformation in water, and displayed parallel self-orientation behavior. AF1 could self-assemble into well defined hollow nanofibers with diameters less than 100 nm and lengths of tens of micrometers in dilute solution, supported by scanning electron microscopy (SEM). Moreover, AF1 in the disulfonated tetraphenylethene (TPE-SO3Na) aqueous solution exhibited strong luminescence, indicating that the TPE-SO3Na molecules without luminescence in water were trapped in the cavities of the hollow nanofibers through hydrophobic interactions, leading to the aggregation-induced emission (AIE). The nanofibers were composed of relatively hydrophobic inner-walls and hydrophilic shells in water. Interestingly, SEM and polarized light microscopy verified that the nanofibers fused to form an ordered architecture of lamella and then tended to curl into hollow fibers in relatively concentrated solution. The hollow fibers exhibited excellent tensile strength, biocompatibility, organic solvent resistance and birefringence. A schematic model was proposed to describe the construction of the hollow fibers via the hierarchical self-assembly process. The new materials would have potential applications such as drug release as a new class of fibrous carrier, indicators with fluorescence to detect cell growth in cell transplantation, and biomolecular recognition (e.g., DNA).

Construction of selenium nanoparticles/β-glucan composites for enhancement of the antitumor activity

We report on a green procedure for the stabilization of selenium nanoparticles (SeNPs) by a naturally occurring β-glucan with triple helical conformation known as Lentinan (t-LNT) in water after denaturing into single chains (s-LNT) at 140 °C. The results demonstrated that the s-LNT can interact with SeNPs through Se-O-H interaction. Transmission electron microscopy (TEM), energy dispersive X-ray (EDX) spectra, UV/vis, X-ray diffraction (XRD) and dynamic light scattering (DLS) showed that s-LNT coated SeNPs to form a stable nano-composite Se/s-LNT, leading to good dispersion of SeNPs. Especially, the as-prepared Se/s-LNT composite in the solution could remain homogeneous and translucent for 30 days without any precipitates. Different size distribution of SeNPs was prepared by simply controlling the concentrations of selenite sodium and the corresponding reducing agent ascorbic acid. The size effect of SeNPs on anti-tumor activity was revealed that the SeNPs with more evenly particle size distribution show the higher anticancer activity.

β-Glucan from Saccharomyces cerevisiae reduces lipopolysaccharide-induced inflammatory responses in RAW264.7 macrophages.

BACKGROUND β-Glucans obtained from fungi, such as bakers yeast (Saccharomyces cerevisiae)-derived β-glucan (BBG), potently activate macrophages through nuclear factor κB (NFκB) translocation and activation of its signaling pathways. The mechanisms by which β-glucans activate these signaling pathways differ from that of lipopolysaccharide (LPS). However, the effects of β-glucans on LPS-induced inflammatory responses are poorly understood. Here, we examined the effects of BBG on LPS-induced inflammatory responses in RAW264.7 mouse macrophages. METHODS We explored the actions of BBG in RAW264.7 macrophages. RESULTS BBG inhibited LPS-stimulated nitric oxide (NO) production in RAW264.7 macrophages by 35-70% at concentrations of 120-200μg/ml. BBG also suppressed mRNA and protein expression of LPS-induced inducible NO synthase (iNOS) and mitogen-activated protein kinase phosphorylation, but not NFκB activation. By contrast, a neutralizing antibody against dectin-1, a β-glucan receptor, did not affect BBG-mediated inhibition of NO production. Meanwhile, BBG suppressed Pam3CSK-induced NO production. Moreover, BBG suppressed LPS-induced production of pro-and anti-inflammatory cytokines, including interleukin (IL)-1α, IL-1ra, and IL-27. CONCLUSIONS Our results indicate that BBG is a powerful inhibitor of LPS-induced NO production by downregulating iNOS expression. The mechanism involves inactivation of mitogen-activated protein kinase and TLR2 pathway, but is independent of dectin-1. GENERAL SIGNIFICANCE BBG might be useful as a novel agent for the chemoprevention of inflammatory diseases.

Anti-tumor effect of β-glucan from Lentinus edodes and the underlying mechanism.

β-Glucans are well known for its various bioactivities, but the underlying mechanism has not been fully understood. This study focuses on the anti-tumor effect and the potential mechanism of a branched β-(1, 3)-glucan (LNT) extracted from Lentinus edodes. The in vivo data indicated that LNT showed a profound inhibition ratio of ~75% against S-180 tumor growth, even significantly higher than the positive control of Cytoxan (~54%). Interestingly, LNT sharply promoted immune cells accumulation into tumors accompanied by cell apoptosis and inhibition of cell proliferation during tumor development. Furthermore, LNT not only up-regulated expressions of the tumor suppressor p53, cell cycle arrestin p21 and pro-apoptotic proteins of Bax and caspase 3/9, but also down-regulated PARP1 and anti-apoptotic protein Bcl-2 expressions in tumor tissues. It was first found that LNT initiated p53-dependent signaling pathway to suppress cell proliferation in vitro, and the caspase-dependent pathway to induce cell apoptosis in vivo. The underlying anti-tumor mechanism was proposed that LNT activated immune responses to induce cell apoptosis through caspase 3-dependent signaling pathway and to inhibit cell proliferation possibly via p53-dependent signaling pathway in vivo. Besides, LNT inhibited angiogenesis by suppressing VEGF expression, leading to slow progression of tumors.

Synthesis and stabilization of gold nanoparticles induced by denaturation and renaturation of triple helical β-glucan in water.

We report on a green procedure for the synthesis and stabilization of gold nanoparticles (AuNPs) from chlorauric acid (HAuCl4) with the use of a β-glucan known as Lentinan (LNT) without external reducing or stabilizing agents in aqueous medium. LNT adopted triple helical conformation in water, which was first denatured into single chains (s-LNT) at a high temperature of 140 °C before mixing with HAuCl4. Results from UV-vis absorption spectroscopy, transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) spectra suggested that AuCl4(-) was rapidly reduced to AuNPs by s-LNT. Moreover, the as-prepared AuNPs could be converted into nanobelt, spherical nanoparticles, and nanowire morphology simply by controlling the s-LNT concentration, reaction time, and temperature. In particular, the AuNPs nanowire was confirmed as the most stable shape in water, which was predominately ascribed to the hydrophobic cavity in the helical center of the renatured triple helical LNT (r-LNT) from s-LNT. Namely, AuNPs were entrapped in the hydrophobic cavity of r-LNT to form nanowire with an outer layer of water-soluble r-LNT, leading to stable dispersion of AuNPs. All the data demonstrated that the β-glucan of s-LNT can be used as a reducing and stabilizing agent to synthesize and disperse AuNPs in water. The whole process of reduction and stabilization was free of organic solvent and thus very safe, which is important for the potential application of AuNPs in biotechnology and biomedicine.